Method for producing polymers

ABSTRACT

The invention relates to a method for producing polymers, in particular synthetic nucleic acid double strands of optional sequence, comprising the steps: (a) provision of a support having a surface area which contains a plurality of individual reaction areas, (b) location-resolved synthesis of nucleic acid fragments having in each case different base sequences in several of the individual reaction areas, and (c) detachment of the nucleic acid fragments from individual reaction areas.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of application Ser. No.10/455,369 filed on 6 Jun. 2003, which in turn is a divisional ofapplication Ser. No. 09/869,332 filed on 26 Jul. 2001, now U.S. Pat. No.6,586,211, which in turn is a national stage filing under 35 U.S.C. §371of PCT/EP00/01356 filed on 18 Feb. 2000, which claims priority to Germanpatent application No. 199 57 116.3 filed on 26 Nov. 1999, Internationalpatent application No. PCT/EP99/06316 filed on 27 Aug. 1999, Germanpatent application No. 199 40 752.5 filed on 27 Aug. 1999, German patentapplication No. 199 28 843.7 filed on 24 Jun. 1999 and German patentapplication No. 199 07 080.6 filed on 19 Feb. 1999.

The invention relates to a method for producing polymers, in particularsynthetic nucleic acid double strands of optional sequence.

TECHNICAL BACKGROUND OF THE INVENTION

Manipulation and construction of genetic elements such as, for example,gene fragments, whole genes or regulatory regions through thedevelopment of DNA recombination technology, which is often alsoreferred to as genetic engineering, led to a particular need for geneticengineering methods and further development thereof in the areas of genetherapy, molecular medicine (basic research, vector development,vaccines, regeneration, etc.). Important areas of application are alsothe development of active substances, production of active substances inthe context of the development of pharmaceuticals, combinatorialbiosynthesis (antibodies, effectors such as growth factors, neuraltransmitters, etc.), biotechnology (e.g. enzyme design, pharming,biological production methods, bioreactors, etc.), diagnostics(BioChips, receptors/antibodies, enzyme design, etc.) and environmentaltechnology (specialized or custom microorganisms, production processes,cleaning-up, sensors, etc.).

PRIOR ART

Numerous methods, first and foremost enzyme-based methods, allowspecific manipulation of DNA for different purposes.

All of said methods have to use available genetic material. Saidmaterial is, on the one hand, well-defined to a large extent but allows,on the other hand, in a kind of “construction kit system” only a limitedamount of possible combinations of the particular available and slightlymodified elements.

In this connection, completely synthetic DNA has so far played only aminor part in the form of one of these combinatorial elements, with theaid of which specific modifications of the available genetic materialare possible.

The known methods share the large amount of work required, combined witha certain duration of appropriate operations, since the stages ofmolecular biological and in particular genetic experiments such as DNAisolation, manipulation, transfer into suitable target cells,propagation, renewed isolation, etc. usually have to be repeated severaltimes. Many of the operations which come up can only insufficiently beautomated and accelerated so that the corresponding work remainstime-consuming and labor-intensive. For the isolation of genes, whichmust precede functional study and characterization of the gene product,the flow of information is in most cases from isolated RNA (mRNA) viacDNA and appropriate gene libraries via complicated screening methods toa single clone. The desired DNA which has been cloned in said clone isfrequently incomplete, so that further screening processes follow.

Finally, the above-described recombination of DNA fragments has onlylimited flexibility and allows, together with the described amount ofwork required, only few opportunities for optimization. In view of thevariety and complexity in genetics, functional genomics and proteomics,i.e. the study of gene product actions, such optimizations in particularare a bottleneck for the further development of modem biology.

A common method is recombination by enzymatic methods (in vitro): here,DNA elements (isolated genomic DNA, plasmids, amplicons, viral orbacterial genomes, vectors) are first cut into fragments with definedends by appropriate restriction enzymes. Depending on the composition ofthese ends, it is possible to recombine the fragments formed and to linkthem to form larger DNA elements (likewise enzymatically). For DNApropagation purposes, this is frequently carried out in a plasmid actingas cloning vector.

The recombinant DNA normally has to be propagated clonally in suitableorganisms (cloning) and, after this time-consuming step and isolation byappropriate methods, is again available for manipulations such as, forexample, recombinations. However, the restriction enzyme cleavage sitesare a limiting factor in this method: each enzyme recognizes a specificsequence on the (double-stranded) DNA, which is between three and twelvenucleotide bases in length, depending on the particular enzyme, andtherefore, according to statistical distribution, a particular number ofcleavage sites at which the DNA strand is cut is present on each DNAelement. Cutting the treated DNA into defined fragments, which cansubsequently be combined to give the desired sequence, is important forrecombination. Sufficiently different and specific enzymes are availablefor recombination technology up to a limit of 10-30 kilo base pairs(kbp) of the DNA to be cut. In addition, preliminary work and commercialsuppliers provide corresponding vectors which take up the recombinantDNA and allow cloning (and thus propagation and selection). Such vectorscontain suitable cleavage sites for efficient recombination andintegration.

With increasing length of the manipulated DNA, however, the rules ofstatistics give rise to the problem of multiple and unwanted cleavagesites. The statistical average for an enzyme recognition sequence of 6nucleotide bases is one cleavage site per 4000 base pairs (4⁶) and for 8nucleotide bases it is one cleavage site per 65,000 (4⁸) Recombinationusing restriction enzymes therefore is not particularly suitable formanipulating relatively large DNA elements (e.g. viral genomes,chromosomes, etc.).

Recombination by homologous recombination in cells is known, too. Here,if identical sequence sections are present on the elements to berecombined, it is possible to newly assemble and manipulate relativelylarge DNA elements by way of the natural process of homologousrecombination. These recombination events are substantially moreindirect than in the case of the restriction enzyme method and,moreover, more difficult to control. They often give distinctly pooreryields than the above-described recombination using restriction enzymes.

A second substantial disadvantage is restriction to the identicalsequence sections mentioned which, on the one hand, have to be presentin the first place and, on the other hand, are very specific for theparticular system. The specific introduction of appropriate sequencesitself then causes considerable difficulties.

An additional well-known method is the polymerase chain reaction (PCR)which allows enzymatic DNA synthesis (including high multiplication) dueto the bordering regions of the section to be multiplied indicating aDNA replication start by means of short, completely synthetic DNAoligomers (“primers”). For this purpose, however, these flanking regionsmust be known and be specific for the region lying in between. Whenreplicating the strand, however, polymerases also incorporate wrongnucleotides, with a frequency depending on the particular enzyme, sothat there is always the danger of a certain distortion of the startingsequence. For some applications, this gradual distortion can be verydisturbing. During chemical synthesis, sequences such as, for example,the above-described restriction cleavage sites can be incorporated intothe primers. This allows (limited) manipulation of the completesequence. The multiplied region can now be in the region of approx. 30kbp, but most of this DNA molecule is the copy of a DNA already present.

The primers are prepared using automated solid phase synthesis and arewidely available, but the configuration of all automatic synthesizersknown to date leads to the production of amounts of primer DNA(μmol-range reaction mixtures) which are too large and not required forPCR, while the variety in variants remains limited.

Synthetic DNA Elements

Since the pioneering work of Khorana (inter alia in: Shabarova: AdvancedOrganic Chemistry of Nucleic Acids, VCH Weinheim;) in the 1960s,approaches in order to assemble double-stranded DNA with genetic orcoding sequences from chemically synthesized DNA molecules haverepeatedly been described. State of the art here is genetic elements ofup to approx. 2 kbp in length which are synthesized from nucleic acids.Chemical solid phase synthesis of nucleic acids and peptides has beenautomated. Appropriate methods and devices have been described, forexample, in U.S. Pat. No. 4,353,989 and U.S. Pat. No. 5,112,575.

Double-stranded DNA is synthesized from short oligonucleotides accordingto two methods (see Holowachuk et al., PCR Methods and Applications,Cold Spring Harbor Laboratory Press): on the one hand, the completedouble strand is synthesized by synthesizing single-stranded nucleicacids (with suitable sequence), attaching complementary regions byhybridization and linking the molecular backbone by, for example,ligase. On the other hand, there is also the possibility of synthesizingregions overlapping at the edges as single-stranded nucleic acids,attachment by hybridization, filling in the single-stranded regions viaenzymes (polymerases) and linking the backbone.

In both methods, the total length of the genetic element is restrictedto only a few thousand nucleotide bases due to, on the one hand, theexpenditure and production costs of nucleic acids in macroscopic columnsynthesis and, on the other hand, the logistics of nucleic acids beingprepared separately in macroscopic column synthesis and then combined.Thus, the same size range as in DNA recombination technology is covered.

To summarize, the prior art can be described as a procedure in which, inanalogy to logical operations, the available matter (in this casegenetic material in the form of nucleic acids) is studied and combined(recombination). The result of recombination experiments of this kind isthen studied and allows conclusions, inter alia about the elementsemployed and their combined effect. The procedure may therefore bedescribed as (selectively) analytical and combinatorial.

The prior art thus does not allow any systematic studies of anycombinations whatsoever. The modification of the combined elements isalmost impossible. Systematic testing of modifications is impossible.

SUBJECT OF THE INVENTION AND OBJECT ACHIEVED THEREWITH

It is intended to provide a method for directly converting digitalgenetic information (target sequence, databases, etc.) into biochemicalgenetic information (nucleic acids) without making use of nucleic acidfragments already present.

The invention therefore relates to a method for producing polymers, inwhich a plurality of oligomeric building blocks is synthesized on asupport by parallel synthesis steps, is detached from the support and isbrought into contact with one another to synthesize the polymer.Preference is given to synthesizing double-stranded nucleic acidpolymers of at least 300 bp, in particular at least 1000 bp in length.The nucleic acid polymers are preferably selected from genes, geneclusters, chromosomes, viral and bacterial genomes or sections thereof.The oligomeric building blocks used for synthesizing the polymer arepreferably 5-150, particularly preferably 5-30, monomer units in length.In successive steps, it is possible to detach in each case partiallycomplementary oligonucleotide building blocks from the support and tobring them into contact with one another or with the polymerintermediate under hybridization conditions. Further examples ofsuitable polymers are nucleic acid analogs and proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a vertical section of a reaction support 30 which isorthogonal to the microchannels 33 present thereon, which are separatedfrom one another by walls 32. The bottom 31 of the reaction support istransparent. Furthermore, a single-stranded nucleic acid 10 with thedesignation of the 5′ and 3′ ends according to convention is depicteddiagrammatically. These are depicted as 10 a with the 3′ end covalentlybound to the reaction support 30 by solid-phase synthesis. A lightsource matrix 20 with a light source and a controllable illuminationexit facing the reaction support 30 is likewise depicted.

FIG. 2 shows a top view of reaction support 30 with reaction areas 12and the walls 32 between the microchannels 33. The arrows indicate thedirection of flow.

FIG. 3 shows, similar to FIG. 1, a vertical section through the reactionsupport 30, with the single-stranded nucleic acids in the microchannel33 being detached.

FIG. 4 again depicts a top view of the reaction support 30, with thesingle-stranded nucleic acids in the microchannel 33 being detached.

FIG. 5 shows a top view of the arrangement of microchannels with fluidicreaction spaces 50, which contain the individual reaction areas, andreaction chambers, where a part sequence is assembled. In the reactionspace 54 all microchannels within a reaction support are broughttogether. The final synthesis product is assembled there, too, and isremoved through exit 55. The reference numbers 51a and 51b indicate therepresentations of a reaction chamber which are shown in enlarged formin FIG. 6 and FIG. 7 and FIG. 8. The arrows again signal the directionof flow.

FIG. 6 shows an enlarged representation of a reaction chamber 51a aftera microchannel with detached single-stranded nucleic acids.

FIG. 7 shows an enlarged representation of a reaction chamber 51a aftera microchannel with a double-stranded hybrid 60 composed of two attachedcomplementary nucleic acid single strands.

FIG. 8 shows an enlarged representation of a reaction chamber 51b afterbringing together two microchannels with an assembled double-strandednucleic acid hybrid 62, enzyme 63 (e.g. ligases) for the covalentlinkage of the building blocks of the nucleic acid hybrid 85, a linearcovalently linked nucleic acid double strand 65 and a circular closednucleic acid double strand 66 (e.g. vector).

The reference number 64 represents a reaction of the enzymes with thenucleic acid hybrid.

DESCRIPTION OF THE INVENTION

In a particularly preferred embodiment, the invention relates to amethod for producing synthetic DNA of any optional sequence and thus anyknown or novel functional genetic elements which are contained in saidsequence. This method comprises the steps

-   -   (a) provision of a support having a surface area which contains        a plurality of individual reaction areas,    -   (b) location-resolved synthesis of nucleic acid fragments having        in each case different base sequences in several of the        individual reaction areas, and    -   (c) detachment of the nucleic acid fragments from individual        reaction areas.

The base sequences of the nucleic acid fragments synthesized inindividual reaction areas are preferably chosen such that they canassemble to form a nucleic acid double strand hybrid. The nucleic acidfragments can then be detached in step (c) in one or more steps underconditions such that a plurality, i.e. at least some of the detachednucleic acid fragments assemble to form a nucleic acid double strandhybrid. Subsequently, the nucleic acid fragments forming one strand ofthe nucleic acid double strand hybrid can at least partially be linkedcovalently to one another. This may be carried out by enzymatictreatment, for example using ligase, or/and filling in gaps in thestrands using DNA polymerase.

The method comprises within the framework of a modular system thesynthesis of very many individual nucleic acid strands which serve asbuilding blocks and, as a result, a double-stranded nucleic acidsequence which can be more than 100,000 base pairs in length isgenerated, for example in a microfluidic reaction support.

The highly complex synthetic nucleic acid which preferably consists ofDNA is produced according to the method and according to the followingprinciple: first, relatively short DNA strands are synthesized in amultiplicity of reaction areas on a reaction support by in situsynthesis. This may take place, for example, using the supportsdescribed in the patent applications DE 19924 327.1, DE 19940 749.5,PCT/EP99/06316 and PCT/EP99/06317. In this connection, each reactionarea is suitable for the individual and specific synthesis of anindividual given DNA sequence of approx. 10-100 nucleotides in length.These DNA strands form the building blocks for the specific synthesis ofvery long DNA molecules. The fluidic microprocessor used here may carryreaction spaces specially designed for the application.

The DNA synthesis itself is thus carried out by following the automatedsolid phase synthesis but with some novel aspects: the “solid phase” inthis case is an individual reaction area on the surface of the support,for example the wall of the reaction space, i.e. it is not particlesintroduced into the reaction space as is the case in a conventionalsynthesizer. Integration of the synthesis in a microfluidic reactionsupport (e.g. a structure with optionally branched channels and reactionspaces) makes it possible to introduce the reagents and other componentssuch as enzymes.

After synthesis, the synthesized building blocks are detached from saidreaction areas. This detachment process may be carried out location-or/and time-specifically for individual, several or all DNA strands.

In a preferred variant of the method it is provided for a plurality ofreaction areas to be established and utilized within a fluidic space orcompartment so that the DNA strands synthesized therein can be detachedin one operation step and taken away from the compartment whichfluidically connects the reaction areas.

Subsequently, suitable combinations of the detached DNA strands areformed. Single-stranded or/and double-stranded building blocks are thenassembled, for example, within a reaction space which may comprise oneor more reaction areas for the synthesis. Expediently, the sequence ofthe individual building blocks is chosen such that, when bringing theindividual building blocks into contact with one another, regionscomplementary to one another are available at the two ends broughttogether, in order to make possible specific attachment of further DNAstrands by hybridizing said regions. As a result, longer DNA hybrids areformed. The phosphorus diester backbone of these DNA hybrids may becovalently closed, for example by ligases, and possible gaps in thedouble strand may be filled in in a known manner enzymatically by meansof polymerases. Single-stranded regions which may be present may befilled in by enzymes (e.g. Klenow fragment) with the addition ofsuitable nucleotides. Thus longer DNA molecules are formed. By bringingtogether clusters of DNA strands synthesized in this way within reactionspaces it is in turn possible to generate longer part sequences of thefinal DNA molecule. This may be done in stages, and the part sequencesare put together to give ever longer DNA molecules. In this way it ispossible to generate very long DNA sequences as completely syntheticmolecules of more than 100,000 base pairs in length.

The amount of individual building blocks which is required for a longsynthetic DNA molecule is dealt with in the reaction support by parallelsynthesis of the building blocks in a location- or/and time-resolvedsynthesis process. In the preferred embodiment, this parallel synthesisis carried out by light-dependent location- or/and time-resolved DNAsynthesis in a fluidic microprocessor which is also described in thepatent applications DE 199 24 327.1, DE 199 40 749.5, PCT/EP99/06316 andPCT/EP99/06317.

The miniaturized reaction support here causes a reduction in the amountof starting substances by at least a factor of 1000 compared with aconventional DNA synthesizer. At the same time, an extremely high numberof nucleic acid double strands of defined sequence is produced. Only inthis way is it possible to generate a very large variety of individualbuilding blocks, which is required for the synthesis of long DNAmolecules, by using an economically sensible amount of resources. Thesynthesis of a sequence of 100,000 base pairs, composed of overlappingbuilding blocks of 20 nucleotides in length, requires 10,000 individualbuilding blocks. This can be achieved using appropriately miniaturizedequipment in a highly parallel synthesis process.

For efficient processing of genetic molecules and systematic inclusionof all possible variants it is necessary to produce the individualbuilding block sequences in a flexible and economic way. This isachieved by the method preferably by using a programmable light sourcematrix for the light-dependent location- or/and time-resolved in situsynthesis of the DNA strands, which in turn can be used as buildingblocks for the synthesis of longer DNA strands. This flexible synthesisallows free programming of the individual building block sequences andthus also generation of any variants of the part sequences or the finalsequence, without the need for substantial modifications of systemcomponents (hardware). This programmed synthesis of the building blocksand thus the final synthesis products makes it possible tosystematically process the variety of genetic elements. At the sametime, the use of computer-controlled programmable synthesis allowsautomation of the entire process including communication withappropriate databases.

With a given target sequence, the sequence of the individual buildingblocks can be selected efficiently, taking into account biochemical andfunctional parameters. After putting in the target sequence (e.g. from adatabase), an algorithm makes out suitable overlapping regions.Depending on the task, different amounts of target sequences can beproduced, either within one reaction support or spread over a pluralityof reaction supports. The hybridization conditions for formation of thehybrids, such as, for example, temperature, salt concentrations, etc.,are adjusted to the available overlap regions by an appropriatealgorithm. Thus, maximum attachment specificity is ensured. In a fullyautomatic version, it is also possible to take target sequence datadirectly from public or private databases and convert them intoappropriate target sequences. The products generated may in turn beintroduced optionally into appropriately automated processes, forexample into cloning in suitable target cells.

Synthesis in stages by synthesizing the individual DNA strands inreaction areas within enclosed reaction spaces also allows the synthesisof difficult sequences, for example those with internal repeats ofsequence sections, which occur, for example, in retroviruses andcorresponding retroviral vectors. The controlled detachment of buildingblocks within the fluidic reaction spaces makes a synthesis of anysequence possible, without problems being generated by assigning theoverlapping regions on the individual building blocks.

The high quality requirements necessary for synthesizing very long DNAmolecules can be met inter alia by using real-time quality control. Thiscomprises monitoring the location-resolved building block synthesis,likewise detachment and assembly up to production of the final sequence.Then all processes take place in a transparent reaction support. Inaddition, the possibility to follow reactions and fluidic processes intransmitted light mode, for example by CCD detection, is created.

The miniaturized reaction support is preferably designed such that adetachment process is possible in the individual reaction spaces andthus the DNA strands synthesized on the reaction areas located withinthese reaction spaces are detached individually or in clusters. In asuitable embodiment of the reaction support it is possible to assemblethe building blocks in reaction spaces in a process in stages and alsoto remove building blocks, part sequences or the final product or elseto sort or fractionate the molecules.

The target sequence, after its completion, may be introduced asintegrated genetic element into cells by transfer and thereby be clonedand studied in functional studies. Another possibility is to firstlyfurther purify or analyze the synthesis product, a possible example ofsaid analysis being sequencing. The sequencing process may also beinitiated by direct coupling using an appropriate apparatus, for exampleusing a device described in the patent applications DE 199 24 327.1, DE199 40 749.5, PCT/EP99/06316 and PCT/EP99/06317 for the integratedsynthesis and analysis of polymers. It is likewise conceivable toisolate and analyze the generated target sequences after cloning.

The method of the invention provides via the integrated genetic elementsgenerated therewith a tool which, for the further development ofmolecular biology, includes biological variety in a systematic process.The generation of DNA molecules with desired genetic information is thusno longer the bottleneck of molecular biological work, since allmolecules, from small plasmids via complex vectors to mini chromosomes,can be generated synthetically and are available for further work.

The production method allows generation of numerous different nucleicacids and thus a systematic approach for questions concerning regulatoryelements, DNA binding sites for regulators, signal cascades, receptors,effect and interactions of growth factors, etc.

The integration of genetic elements into a fully synthetic completenucleic acid makes it possible to further utilize known genetic toolssuch as plasmids and vectors and thus to build on the relevantexperience. On the other hand, this experience will change rapidly as aresult of the intended optimization of available vectors, etc. Themechanisms which, for example, make a plasmid suitable for propagationin a particular cell type can be studied efficiently for the first timeon the basis of the method of the invention.

This efficient study of large numbers of variants makes it possible todetect the entire combination space of genetic elements. Thus, inaddition to the at the moment rapidly developing highly parallelanalysis (inter alia on DNA arrays or DNA chips), the programmedsynthesis of integrated genetic elements is created as a secondimportant element. Only both elements together can form the foundationof an efficient molecular biology.

The programmed synthesis of appropriate DNA molecules makes possible notonly random composition of the coding sequences and functional elementsbut also adaptation of the intermediate regions. This may rapidly leadto minimal vectors and minimal genomes, whose small size in turngenerates advantages. As a result, transfer vehicles such as, forexample, viral vectors can be made more efficient, for example whenusing retroviral or adenoviral vectors.

In addition to the combination of known genetic sequences, it ispossible to develop novel genetic elements which can build on thefunction of available elements. Especially for such developmental work,the flexibility of the system is of enormous value.

The synthetic DNA molecules are in each stage of the development of themethod described here fully compatible with the available recombinationtechnology. For “traditional” molecular biological applications it isalso possible to provide integrated genetic elements, for example byappropriate vectors. Incorporation of appropriate cleavage sites even ofenzymes little used so far is not a limiting factor for integratedgenetic elements.

Improvements in Comparison with Prior Art

This method makes it possible to integrate all desired functionalelements as “genetic modules” such as, for example, genes, parts ofgenes, regulatory elements, viral packaging signals, etc. into thesynthesized nucleic acid molecule as carrier of genetic information.This integration leads to inter alia the following advantages:

It is possible to develop therewith extremely functionally integratedDNA molecules, unnecessary DNA regions being removed (minimal genes,minimal genomes).

The free combination of the genetic elements and also modifications ofthe sequence such as, for example, for adaptation to the expressingorganism or cell type (codon usage) are made possible as well asmodifications of the sequence for optimizing functional geneticparameters such as, for example, gene regulation.

Modifications of the sequence for optimizing functional parameters ofthe transcript, for example splicing, regulation at the mRNA level,regulation at the translation level, and, moreover, the optimization offunctional parameters of the gene product, such as, for example, theamino acid sequence (e.g. antibodies, growth factors, receptors,channels, pores, transporters, etc.) are likewise made possible.

On the whole, the system created by the method is extremely flexible andallows in a manner previously not available the programmed production ofgenetic material under greatly reduced amounts of time, materials andwork needed.

Using the available methods, it has been almost impossible tospecifically manipulate relatively large DNA molecules of severalhundred kbp, such as chromosomes for example. Even more complex (i.e.larger) viral genomes of more than 30 kbp (e.g. adenoviruses) aredifficult to handle and to manipulate using the classical methods ofgene technology.

The method of the invention leads to a considerable shortening up to thelast stage of cloning a gene: the gene or the genes are synthesized asDNA molecule and then (after suitable preparation such as purification,etc.) introduced directly into target cells and the result is studied.The multi-stage cloning process which is mostly carried out inmicroorganisms such as E. coli (e.g. DNA isolation, purification,analysis, recombination, cloning in bacteria, isolation, analysis, etc.)is thus reduced to the last transfer of the DNA molecule into the finaleffector cells. For synthetically produced genes or gene fragmentsclonal propagation in an intermediate host (usually E. coli) is nolonger required. This avoids the danger of the gene product destined forthe target cell exerting a toxic action on the intermediate host. Thisis distinctly different from the toxicity of some gene products, which,when using classical plasmid vectors, frequently leads to considerableproblems for cloning of the appropriate nucleic acid fragments.

Another considerable improvement is the reduction in time and thereduction in operational steps to after the sequencing of geneticmaterial, with potential genes found being verified as such and cloned.Normally, after finding interesting patterns, which are possible openreading frames (ORF), probes are used (e.g. by means of PCR) to searchin cDNA libraries for appropriate clones which, however, need notcontain the whole sequence of the mRNA originally used in theirproduction. In other methods, an expression gene library is searched bymeans of an antibody (screening). Both methods can be shortened verysubstantially using the method of the invention: if a gene sequencedetermined “in silico” is present (i.e. after detection of anappropriate pattern in a DNA sequence by the computer) or after decodinga protein sequence, an appropriate vector with the sequence or variantsthereof can be generated directly via programmed synthesis of anintegrated genetic element and introduced into suitable target cells.

The synthesis taking place in this way of DNA molecules of up to several100 kbp allows the direct complete synthesis of viral genomes, forexample adenoviruses. These are an important tool in basic research(inter alia gene therapy) but, due to the size of their genome (approx.40 kbp), are difficult to handle using classical genetic engineeringmethods. As a result, the rapid and economic generation of variants foroptimization in particular is greatly limited. This limitation isremoved by the method of the invention.

The method leads to integration of the synthesis, detachment ofsynthesis products and assembly to a DNA molecule being carried out inone system. Using production methods of microsystem technology, it ispossible to integrate all necessary functions and process steps up tothe purification of the final product in a miniaturized reactionsupport. These may be synthesis areas, detachment areas (clusters),reaction spaces, feeding channels, valves, pumps, concentrators,fractionation areas, etc.

Plasmids and expression vectors may be prepared directly for sequencedproteins or corresponding part sequences and the products may beanalyzed biochemically and functionally, for example by using suitableregulatory elements. This omits the search for clones in a gene library.Correspondingly, ORFs from sequencing work (e.g. Human Genome Project)can be programmed directly into appropriate vectors and be combined withdesired genetic elements. An identification of clones, for example bycomplicated screening of cDNA libraries, is removed. Thus, the flow ofinformation from sequence analysis to function analysis has been greatlyreduced, because on the same day on which an ORF is present in thecomputer due to analysis of primary data, an appropriate vectorincluding the putative gene can be synthesized and made available.

Compared with conventional solid-phase synthesis for obtaining syntheticDNA, the method according to the invention is distinguished by a smallamount of material needed. In order to produce thousands of differentbuilding blocks for generating a complex integrated genetic element ofseveral 100,000 kbp in length, in an appropriately parallelized formatand with appropriate miniaturization (see exemplary embodiments), amicrofluidic system needs markedly fewer starting substances for anindividual DNA oligomer than a conventional solid-phase synthesisapparatus (when using a single column). Here, microliters compare withthe consumption of milliliters, i.e. a factor of 1000.

Taking into account the newest findings in immunology, the presentedmethod allows an extremely efficient and rapid vaccine design (DNAvaccines). Exemplary Embodiments

To carry out the method, the present invention requires the provision ofa large number of nucleic acid molecules, usually DNA, whose sequencecan be freely determined. These building blocks must have virtually 100%identical sequences within one building block species (analogously tothe synthesis performance of conventional synthesizers). Only highlyparallel synthesis methods are suitable for generating the requiredvariance. In order for the system to be able to work flexibly and,despite the necessary multiplicity of different building blocks to besynthesized, to require as little space and as few reagents as possible,the method is preferably carried out in a microfluidic system withinwhich the individual sequences are produced in a determinable form. Twotypes of programmed synthesis are suitable for systems of this kind,which are also described in the patent applications DE 199 24 327.1, DE199 40 749.5, PCT/EP99/06316 and PCT/EP99/06317: these are first thesynthesis by programmable fluidic individualization of the reactionareas and, secondly, the synthesis by programmable light-dependentindividualization of the reaction areas.

In both variants, synthesis is carried out in a microfluidic reactionsupport. The design of this reaction support may provide in the systemfor the bringing together in stages the detached synthesis products,i.e. building blocks, by collecting the nucleic acid strands, afterdetaching them, in appropriate reaction areas and the assembly takingplace there. Groups of such assembly areas may then for their part bebrought into contact again with one another so that during the course ofa more or less long cascade the final synthesis products are produced:genetic information carriers in the form of DNA molecules. The followingvariants are suitable here:

Either synthesis, detachment and assembly are carried outchronologically but spatially integrated in a microfluidic reactionsupport or synthesis, detachment and assembly are carried out partiallyin parallel in one or more microfluidic reaction supports. It isfurthermore possible that the microfluidic reaction support containsonly reaction areas for the programmed synthesis and that subsequentlydetachment and elution into a reaction vessel for the assembly arecarried out.

In the case of very large DNA molecules, synthesis, detachment andassembly can be supplemented by condensation strategies which preventbreak-up of the molecules. This includes, for example, the use ofhistones (nuclear proteins which make condensation of the chromosomes inthe nucleus possible in eukaryotes), the use of topoisomerases (enzymesfor twisting DNA in eukaryotes and prokaryotes) or the addition of otherDNA-binding, stabilizing and condensing agents or proteins. Depending onthe design of the reaction support, this may take place by integratingthe condensation reaction in another reaction chamber provided thereforor by addition during the combination and assembly in stages of thebuilding blocks.

The free choice of sequence is of essential importance for thecontrolled and efficient building block assembly in stages to the finalproduct. For the choice of overlapping complementary ends influences thespecificity of the assembly and the overall biochemical conditions (saltconcentration, temperature, etc.). When providing a sequence for thegene of interest and after automatic or manual selection of the othergenetic elements (regulatory regions, resistance genes for cloning,propagation signals, etc.) for determination of the final product (e.g.a plasmid vector), the provided sequence is fragmented into suitablebuilding blocks which are then synthesized in the required number ofreaction supports. The fragments or their overlap regions to behybridized are chosen such that the conditions for hybridizing are assimilar as possible (inter alia GC:AT ratio, melting points, etc.).

Further extension of the system provides for elements for purificationand isolation of the product forming, which are likewise designed bymicrofluidics or microsystem technology. Said elements may be, forexample, methods in which the final double-stranded DNA after itssynthesis using fluorescent synthons must have a particular totalfluorescence. When using proteins with condensing action, theseproteins, where appropriate, may also carry a fluorescent label which ispreferably detectable separately (reference signal). It is then possibleto sort the mixture of final reaction product in the reaction supportstructures according to fluorescence (see Chou et al., Proceedings ofthe National Academy of Science PNAS 96:11-13, 1999). Thus a sufficientquality is achieved in order to directly provide a product for furtherwork.

Information from sequencing projects, which is present in databases, maybe studied for genes fully automatically (computer-assisted). Identifiedor putative genes (ORFs) are converted into completely synthetic DNAwhich may contain, where appropriate, regulatory and other geneticelements which seem suitable, so that, for example, one or more vectorsare generated. The product is either made available (e.g. as pure DNA)or directly introduced to functional studies, inter alia by transferinto suitable target cells. The information may come from publicdatabases, from work of decentralized users or from other sources, forexample the method described in the patent applications DE 199 24 327.1and DE 199 40 749.5.

It may be of interest that a variance of randomized sequence occurs at aparticular site or sites of the target sequence. An example is thetesting of variants of a binding site into which, for example over anarea of 20 amino acids, i.e. 60 nucleotides, random variations ofnucleotides were incorporated. This may take place in an embodiment inthat during the synthesis process, after activating a reaction area, amixture of synthons is added so that all added synthons can hybridize ina statistically distributed manner. A modification of this process mayprovide for DNA building blocks of different length to be used at aparticular position of the target sequence, for example by producingdifferent building blocks on different reaction areas, which show thesame sequence for overlapping and hybridization.

1. Method for synthesizing a minimal genome or a section thereof,characterized in that a plurality of oligomeric building blocks issynthesized on a support by parallel synthesis steps, is detached fromthe support and is brought into contact with one another to synthesizethe minimal genome or section thereof.
 2. Method according to claim 1characterized in that nucleic acid polymers selected from the groupconsisting of genes, minimal genes, gene clusters, chromosomes orsections thereof are synthesized and are brought into contact with oneanother to synthesize the minimal genome or section thereof.
 3. Methodaccording to claim 1 characterized in that the minimal genome is a viralor bacterial genome.
 4. Method according to claim 1 characterized inthat a double-stranded nucleic acid polymer with a length at least100,000 bp is synthesized.
 5. Method according to claim 1 characterizedin that the oligomeric building blocks are from 5 to 150, preferably 5to 30 monomer units in length.
 6. Method according to claim 1characterized in that in successive steps in each case partiallycomplementary oligonucleotide building blocks are detached from thesupport and are brought into contact with one another or with thepolymer intermediate under hybridization conditions.
 7. Method accordingto claim 1 for producing synthetic nucleic acid double strands,comprising the steps: (a) provision of a support having a surface areawhich contains a plurality of individual reaction areas, (b)location-resolved synthesis of nucleic acid fragments having in eachcase different base sequences in several of the individual reactionareas, and (c) detachment of the nucleic acid fragments from individualreaction areas.
 8. Method according to claim 7 characterized in that thebase sequences of the nucleic acid fragments synthesized in individualreaction areas are chosen such that they can assemble to form a nucleicacid double strand hybrid.
 9. Method according to claim 7 characterizedin that the nucleic acid fragments according to step (c) are detached inone or more steps under conditions such that a plurality of the detachednucleic acid fragments assemble to form a nucleic acid double strandhybrid.
 10. Method according to claim 9 characterized in that severalnucleic acid fragments which form one strand of the nucleic acid doublestrand hybrid are linked covalently to one another.
 11. Method accordingto claim 10 characterized in that the covalent linking includestreatment with ligase or/and filing in gaps in the strands using DNApolymerase.
 12. Method according to claim 7 characterized in that thesequence comprises at one or more positions recognition sequences forspecific interaction with molecules such as proteins, nucleic acids,peptides, pharmaceuticals, saccharides, lipids, hormones, or/and organiccompounds.
 13. Method according to claim 7 characterized in that thesequence of the nucleic acid double strands is a naturally occurringsequence, a not naturally occurring sequence or a combination of thesetwo.
 14. Method according to claim 7 characterized in that the sequenceis taken from a database, a sequencing experiment or a device for theintegrated synthesis and analysis of polymers.
 15. Method according toclaim 1 characterized in that the oligomeric building blocks aresynthesized by location- or/and time-resolved illumination by means of aprogrammable light source matrix.
 16. Method according to claim 1characterized in that a location- or/and time-resolved synthesis of theoligomeric building blocks takes place in a microfluidic reactionsupport having one or more fluidic reaction compartments and one or morereaction areas within a fluidic reaction compartment.
 17. Methodaccording to claim 1 characterized in that the synthesis building blockscontain nucleotides occurring in nature, modified nucleotides ormixtures thereof
 18. Method according to claim 1 characterized in thatmodified synthesis building blocks are used for labelling and subsequentdetection of the assembled nucleic acid double strands.
 19. Methodaccording to claim 18 characterized in that the labelling groups usedare molecules which are to be detected in a light-dependent manner. 20.Use of a nucleic acid double strand produced according to the methodaccording to claim 1 for therapeutic or pharmacological purposes. 21.Use of a nucleic acid double strand produced according to the methodaccording to claim 1 for diagnostic purposes.
 22. Use according to claim20 comprising direct application to the intended purpose.
 23. Useaccording to claim 21 comprising direct application to the intendedpurpose.
 24. Use according to claim 20 comprising a conversion ineffector cells.
 25. Use according to claim 21 comprising a conversion ineffector cells.
 26. Use of a nucleic acid double strand producedaccording to the method according to claim 1, where said nucleic aciddouble strand is stabilized, condensed or/and topologically manipulatedduring or following the combination and assembly in stages.
 27. Useaccording to claim 26, where stabilization, condensation or/andtopological manipulation is carried out by functional molecules such ashistones or topoisomerases.
 28. Use of a nucleic acid double strandproduced according to the method according to claim 1 as propagatablecloning vector, where the propagatable cloning vector may serve fortranscription, expression of the transcribed sequence, and, whereappropriate, production of expressed gene products in suitable targetcells.